Termites are considered to be one of the most important bioreactors on the planet. Termites have developed cellulose digestion capabilities that allow them to obtain energy and nutrition from nutritionally poor food sources, such as cellulose. Because of their roles in nutrient and carbon cycling in natural ecosystems, termites are insects with tremendous positive global value. However, because of their cellulose feeding/digestion capabilities termites are also globally important as economic pests. As a result of their damage to human structures and commodities, it has recently been estimated that annual termite damage, control and repair costs exceed $20 billion worldwide.
Subterranean termites from the genera Reticulitermes and Coptotermes are among the most destructive species worldwide. In particular, the U.S. Eastern subterranean termite R. flavipes and its European synonym R. santonensis are among the most important pest termites on their respective continents. Millions of gallons of soil termiticides are applied every year in the US and Europe to protect from subterranean termite damage. A more environmentally-friendly method of termite control is through baiting, which involves recruiting termites to feed on cellulosic substrates impregnated with low concentrations of slow-acting chemical insecticides. A major drawback to baiting, however, is its lack of speed in termite colony elimination. In order to reduce reliance on non-specific soil termiticides and increase speed of colony elimination through baiting, there is a need for effective, faster-acting termite bait active ingredients. The instant invention provides a significant new method of termite control utilizing RNA interference techniques.
The cellulases are a family of enzymes that hydrolyze the beta 1,4, linkages of cellulose and permit this conversion of cellulose into energy sources. They appear to have endogenous cellulases, as well as celluloses that arise from symbionts that live in the termites' guts. The biologically mediated degradation of cellulose requires several functional types of cellulases, mainly endoglucanases that act on longer cellulose chains, and exoglucanases and beta glucosidases.
Prior research has identified various genes in the termite R. flavipes called Cell-1,2,3, and 4 that code for cellulases. Cell 1 and Cell 2 are endoglucanases, while Cell 3 and Cell 4 are exoglucanases.
Good to excellent inhibition of exoglucanases and beta glucosidase activity has been observed in in vitro enzyme assays, however, the inhibitors cellobioimidazole (CBI) and fluromethyl cellobiose (FMCB) caused only moderate termite mortality in bioassays. This lack of toxicity suggests that other upstream enzymes that act on longer cellulose chains may be more susceptible target sites for novel cellulase inhibitors, and thus in the instant invention, using RNA interference to create endoglucanase inhibition is addressed as a means for termite population control.
Termites are the only social insect group that undergoes incomplete metamorphosis. Worker termites may differentiate into reproductive or soldier termites, with resultant different behaviors. Worker termites engage in foraging, tunneling and brood tending, the reproductives produce offspring, and the soldiers engage in colony defense. Hexamerin proteins are part of a mechanism that maintains high worker proportions in termite colonies. This mechanism allows termite colonies to retain high proportions of altruistic worker members, such proportion apparently enhancing colony fitness. The morphogenesis of workers to pre-solider and soldier termites (see FIG. 1 and FIG. 5a) is induced by a 2-5 fold increase of the insect juvenile hormone (JH). Hexamerin proteins have well defined roles as JH binding proteins.
Using RNA interference techniques the instant invention demonstrates that silencing the Hex-1 and Hex-2 hexamerin genes increases pre-soldier formation and can be utilized as a means of decreasing the proportion of worker termites in the colony and consequently negatively impacting termite colony fitness. Additionally, targeting other genes that are part of juvenile hormone (JH) signaling is another aspect of the instant invention. Here, genes include the BTP/POZ transcriptional regulator “broad”, the “farnesoic acid methyl transferase-2” gene which encodes an enzyme that is part of the JH biosynthetic pathway, two genes encoding cytochrome P450 enzymes named “Cyp15-1” and “Cyp15-2” that participate in JH biosynthesis and degradation, and two genes encoding vitellogenin proteins that dramatically increase in expression during JH-dependent caste differentiation.
With the ever-pressing demand for termite control compositions that are environmentally safe and effective in preventing termite infestation, researchers are pursuing a number of strategies to overcome problems of prior compositions.
Among the various methods and uses of RNA interference reported in the patent literature are the following.
U.S. Patent Publication 2007/0199100 to Michaeli; Shulamit, et al., shows feeding on plant cytoplasm, including insects, nematodes and fungi, plants engineered to produce small interfering RNAs (siRNAs) capable of silencing parasite specific gene; parasite gene is stage-specific gene, gene involved in essential, early developmental stages of parasite, the plant; uses T7 polymerase recognition sites [0232]; [primers used at the 5′end of gene [0247]; per amplification [0249].
U.S. Patent Publication 2007/0192903 to Heck; Gregory, et al., shows transgenic plant cells, plants and seeds containing modified suppression elements, and useful derivatives of transgenic plant cells, plants/seeds, such as food/feed products; suppression elements any suppression element that when transcribed in eukaryotic cell, results in silencing the target gene; suppression element can be transcribable DNA of any length & 19 to 27 nucleotides (for example 19, 20, 21, 22, 23, or 24 nucleotides) for every target gene suppressed, [0025] and may include siRNA [0034]; plant cells, [0045]; silenced during transcription [0062]; suppression measured by resistance to pest [0064]; 100 base pair fragment amplified by PCR with appropriate primers to produce an antisense template and sense template; sense and antisense reactions mixed, heated to 75 degrees Celsius, cool to room temperature; resulting in annealed 100 base pair double-stranded RNA product; purified with e MEGAscript™ RNAi Kit (Ambion, CAT #1626); produce a 100 base pair dsRNA product; tested with same WCR larval bioassay; fed to WCR larvae at 0.2 ppm, 100 bp dsRNA suppression element induced 100% mortality (FIG. 1); control (double-stranded RNA derived from 108 base pairs of vector sequence) caused no mortality at same feeding concentration [0108].
U.S. Patent Publication No. 2007/0130653 to Baulcombe; David, et al., shows siRNA, coexpressed in cells at appropriate developmental stage provide silencing of final target; final target may be a gene in a second organism, such as a plant pest, that feeds upon the organism containing the chimeric gene or genes.
U.S. Patent Publication 2007/0011775 to Allen; Edwards, et al., shows dsRNA for silencing genes [0008]; aptamer binds to tissue/cell/cell component such as gut of a pest of plant in which RNA is transcribed [0110].
U.S. Patent Publication 2006/0288448 to Abad; Andre R., et al., shows compositions of novel nucleic acid molecules; nucleotide sequences encoding modified insect protoxins provide for efficient processing into active toxins within the gut of the insect pest that feeds on plant host expressing modified insect protoxin; nucleotide sequences that encode modified insect protoxins that have proteolytic activation site engineered with cleavage site sensitive to insect gut protease; cleavage of modified insect protoxin by an insect gut protease produces an active insect toxin in the insect gut; [0015] express the gene from an inducible promotor, particularly from a pathogen-inducible promoter; promoters include those from pathogenesis-related proteins which are induced following infection; e.g. beta-1, 2-gluvanase, chitinase [0089].
U.S. Patent Publication 2006/0272049 to Waterhouse; Peter Michael, et al., shows genes expressed in cells of plant sap-sucking insect gut tissue/midgut are targeted; genes involved in gut cell metabolism/growth/differentiation [0067].
U.S. Patent Publication 2004/0133943 to Plaetinck; Geert, et al., shows alleviating pest infestation of plants, when the pest feeds on plants it will digest expressed dsRNA in plant inhibiting expression of DNA within pest which is critical for its growth, survival, proliferation or reproduction [016]; root will produce RNA/dsRNA from introduced piece of DNA when root specific promoter is utilized; nematode feeds on plant, RNA and/or dsRNA will be consumed or ingested by the nematode; RNA and/or dsRNA can enter the cells of the nematode and perform its inhibitory action on the target DNA1 nature of cloned DNA piece of worm, nematode and perform its inhibitory action on the target DNA; nature of cloned DNA piece of worm, nematode will not be able to survive, to eat, proliferate, preventing the animal of feeding longer on the plant, and thus protecting the plant [0124]; T7 polymerase encoding with PCR, [0127], RNAi phenotype lines [0138].
U.S. Patent Publication 2004/0098761 to Trick; Harold N., et al., shows transgenic plant expressing a heterologous double stranded nematode RNA sequence at level such that nematodes ingesting said heterologous double stranded nematode RNA sequences exhibit decreased proliferation as compared to nematodes feeding on non-transgenic plants [0012]; includes woody plants [0034]; uses RNAi [01116]; use of RNA interference (RNAi) down regulate expression of genes needed for nematode.
U.S. Patent Publication 2004/0018999 to Beach; David, et al., shows inhibition of cellulase by dsRNA [0129].
U.S. Patent Publication 2003/0154508 to Stevens; Bruce Russell, et al., shows control of termites [0044]; using double-stranded interfering RNA (RNAi), RNA-mediated interference (RNAa), [0051] to silence ligand-gated ion channels in the insect gut.
U.S. Patent Publication 2003/0150017 to Mesa; Jose Ramon Botella, et al., shows genetic agents are RNAi [0048]; Down-regulation of expression of target gene prevents/retards pathogen growth, development and/or reproduction [0047]; “pathogen” insect, arachnid or other creature capable of infecting or infesting plant [0122]; uses per [0041].
U.S. Patent Publication 2003/0135888 to Zhu; Tong, et al., shows confer insecticidal activity on plant by altering its sterol composition; sterols are obtained by insects from their diet and are used for hormone synthesis and membrane stability; alteration by expression of novel genes; a negative effect on insect growth and/or development; endow the plant with insecticidal activity; lipoxygenase [0350].
U.S. Patent Publication 2003/0106092 to Davis, Eric L., et al., shows nucleic acid construct; cyst and root knot nematode responsive promoter, Nicotiana Ntcel7 (endoglucanase) promoter associated with heterologous nucleic acid segment that encodes a product disruptive of nematode attack.
U.S. Pat. No. 6,506,559 to Fire, Andrew, et al. describes gene-specific inhibition of gene expression by double-stranded ribonucleic acid dsRNA; target gene may be endoglucanase.
Thus, there are several inventions that relate to transgenic plants that express double strand RNA for insect or pathogen control. Only one of the applications (Stevens et al.) relates to using RNA interference (RNAi) for termite control, but at a different target site than the current invention. Andrew Fire et al. discusses RNAi-based pest control, but does not mention invertebrate control by feeding, nor does it teach invertebrate pest control by RNAi interference or double strand RNA (dsRNA) feeding.
The present invention provides a unique approach to controlling termite populations by using RNAi to increase termite mortality by inhibiting endoglucanases, hexamerins, “broad” BTB/POZ transcription factors, farnesoic acid methyl transferases, cytochrome P450s, and vitellogenins.